WO1999058712A2

WO1999058712A2 - Detection of genes

Info

Publication number: WO1999058712A2
Application number: PCT/DE1999/001423
Authority: WO
Inventors: Fawzy Abdelatty
Original assignee: Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date: 1998-05-12
Filing date: 1999-05-11
Publication date: 1999-11-18
Also published as: DE19820982A1; WO1999058712A3

Abstract

The present invention relates to method for detecting genes and the expression thereof, whereby DNA is amplified with gene-specific primers and the amplified DNA is used for hybridization purposes with a DNA library.

Description

Detection of genes

The present invention relates to a method for the detection of genes and their expression.

For years there has been increasing work to elucidate the genome of living beings, especially animals and humans. In particular, attempts are being made to find genes that control development and metabolic processes. Genes related to diseases are also searched for. Various methods are used for this. However, these processes all suffer from the fact that they are very complex and difficult to reproduce.

The present invention is therefore based on the object of providing a method with which genes can be detected in a simple and reliable manner and, if appropriate, their expression can be determined.

According to the invention, this is achieved by the subject matter in the claims.

The present invention thus relates to a method which comprises the ampiification of DNA and its hybridization with a DNA library, wherein primers are used for the ampiification, the sequences of which are characteristic of genes.

The present invention is based on the knowledge of the applicant that sequences which are highly conserved are present in the transcribed 5 'region of genes, the so-called "CpG islands". The applicant has found these sequences in genes of living beings, in particular animals and humans, viruses and plants. The sequences are given in Table I. They have pallindrome structures and make complete or parts of recognition sequences less common Restriction enzymes. The applicant has also recognized that DNA sequences of genes can be amplified using these sequences as primers and these can be used to detect the genes and their expression.

According to the invention, these findings are used for a method, comprising the following method steps:

(a) Ampiification of a DNA with one to all of the following primers:

5'-GCGCGCNN-3 '5'-GCGGCCGC-3'

5'-CCGCGGNN-3 '

5'-CGCGNNNN-3 '

5'-GGCGCGCC-3 '

5'-GGGCCCNN-3 '5'-CGATCGNN-3'

5'-CGG (A.T) CCGN-3 '

5'-CGGCCGNN-3 '

5'-GGCCGGCC-3 '

5'-ACGCGTNN-3 '5'-GCCGGCNN-3'

5'-GGCGCCNN-3 '

5'-CCNNGGNN-3 '

5'-TCGCGANN-3 '

5'-CGGNCCGN-3 '5'-GTCGACNN-3'

5'-GGCCNNGGCC-3 '

5'-CGCCGGCG-3 '

5'-GCCCGGGC-3 'where N is G or C, and

(b) Hybridizing the amplified DNA with a DNA library.

The term "DNA" includes any DNA from which sections are amplified can, if at least one of the primers of Table I is used. The ampiification can be followed by customary methods, for example via gel electrophoresis (cf. FIG. 1 (A)). In particular, the DNA can be a genomic or an extrachromosomal DNA. It can also be a cDNA. The term "genomic DNA" encompasses both cell DNA and virus DNA which is integrated into the genome. An example of such virus DNA is HPV DNA. Furthermore, the term "extrachromosomal DNA" encompasses any DNA that is not integrated into the genome, which applies to the DNA of lytic viruses. The DNA can come from any cell. These can be kept in culture or freshly isolated from an individual. The cells can be normal or changed or have pathogenic properties. In particular, the cells can be tumor cells. The expression "individual" encompasses any individual, in particular animals or humans and plants. Hybrid animal cells are preferred according to the invention which contain the human X or Y chromosome or a human XY translocation, as a result of which the pseudoautosomal region Xp22.3 has been lost distal to MIC2. Such hybrid animal cells are known (cf. Burk, RD et al., Mol. Cell Biol. 5 (1985), 576-581; Ropers, HH et al., Cytogenet. Cell Genet. 40 (1985), 736.

The term "DNA library" includes any DNA library with which the DNA can hybridize. In particular, the DNA library can be a genomic DNA or a cDNA library. The term "hybridization" indicates hybridization under normal conditions, in particular at 30 ° C. below the melting point of the DNA. The DNA library can be made from DNA from any cell. These can be normal or changed or have pathogenic self-switching. In particular, the cells can be tumor cells. Furthermore, the cells can come from any individual, in particular animals or humans or plants. According to the invention, a DNA library is preferred which contains the human X chromosome in the λ vector Charon 35 (ATCC Cat.No. 57750).

The DNA and the DNA library can come from the same species, in particular from the same individual. They can also be from different individuals of the same species. It may also be beneficial if the DNA and DNA library are from different species.

In a method according to the invention, the primers given in Table I are used to amplify a DNA. The primers can be prepared by conventional methods. Furthermore, the method according to the invention can be carried out under customary amplification and hybridization conditions. Reference is made to example 1.

With the present invention it is possible to detect genes. With the amplification of sequences characteristic of genes and the use of these in hybridization with a DNA library, genes can be assigned to specific clones. The genes can then be further characterized and sequenced using conventional methods. The expression of the genes can also be detected. For this it is advisable to use different cDNAs, e.g. a normal cell and a tumor cell to amplify and hybridize with a cDNA library. Expression differences can thus be determined. Such offers e.g. to find genes related to diseases. Furthermore, the present invention is characterized in that it can be carried out in a simple and reliable manner. In particular, the method according to the invention is generally applicable, i.e. genes from a wide variety of cells or individuals can be detected and their expression determined. The present invention thus makes a major contribution to the elucidation of genomes and their functions.

Brief description of the drawings

1 shows the ampiification of a genomic DNA from the hybrid mouse cell line 578 which contains the human X chromosome (A). Hybridization of the amplified DNA with a genomic DNA library relating to the human X chromosome (ATCC Cat. No. 57750) is shown in (B). 2 shows 10 clones from the DNA library relating to the human X chromosome, which hybridized with the amplified DNA from cell 578. These clones were digested with the restriction enzymes BssHIl, Notl, Sacll and EcoRI, the latter restriction enzyme being used to separate the vector from the inserts. Of the first 3 restriction enzymes, their recognition sequences were used as primers in the amplification of the DNA from the cell line 578. BssHII recognition sequence is found in seven clones, Sacll- in five clones and Notl- in two clones (A, B). Hybridization of the blot of (B) with the amplified DNA from cell line 578 is shown in (C).

FIG. 3 shows 16 clones from the DNA library relating to the human X chromosome, which hybridized with the amplified DNA from the cell line 578. The clones were cleaved with EcoRI, separated on a 0.8% agarose gel, subjected to a blot method and hybridized with the amplified DNA from cell line 578. 15 defined bands can be seen (A). The same filter was washed off and hybridized with the amplified DNA from the hybrid hamster cell 853, which contains the human Y chromosome (B). Four clones also hybridized. Three insert bands from clone X26, including the common XY band, were isolated from the agarose gel and used for hybridization with geπomischen Southern blots, which contained DNA from the cell lines 578, 853 and 815 x175k7 (see description). The common positive band resulted in smear hybridization (data not shown). The smaller band gave an X-specific hybridization (C), while the smallest band a homologous sequence between the

X and Y chromosomes resulted in different hybridization patterns being obtained for both chromosomes (D).

The invention is illustrated by the following example.

Example: Detection of genes from the human X chromosome 1. Ampiification of DNA from hybrid mouse cell line 578

Genomic DNA was isolated from the hybrid mouse cell line 578. 30 ng of the genomic DNA were subjected to an amplification reaction in which the primers BssHIl, Notl and Sacll (see Table I) were used.

The conditions for the amplification reaction were as follows: 1 min at 95 ° C, 1 min at 35 ° C, 1 min at 50 ° C; 30 cycles.

Cold amplification reaction: 100 μl volume: 10 μl (30 ng) genomic DNA, 10 μi (200 ng) primer, 10 μl 2 mM dNTP, 10 μl 10 × reaction buffer, 60 μl H ₂ O, 1 μl (5 units) Taq polymerase.

The amplification products obtained were separated in a 1.2% agarose gel. The products obtained had a size of 100-2000 bp (see FIG. 1 (A)).

Radioactive labeling of the amplification products 100 μl volume:

10 μl cold amplification products, 10 μl (200 ng) primer,

10 μl 10 × C dNTP mixture (200 μM dTTP, dGTP, dATP), 10 μl 10 × reaction buffer, 50 μl H ₂ O,

1 µl (5 units) Taq polymerase, and 10 µl (90 µCi) [α- ^{3 p} ] dCTP.

The amplification reaction was carried out in 10 cycles under the same conditions as for the cold amplification reaction. After completion of the amplification reaction, the labeled amplification products were separated from the unincorporated nucleotides using the "spin column S-400" from Pharmacia. The labeled amplification products were heated for 10 minutes, cooled on ice for 3 minutes and used for hybridization.

2. Hybridization of the amplified DNA with a DNA library

PIaques of 50,000 clones from a genomic DNA library relating to the human X chromosome (ATCC Cat. No. 57750) were transferred to a Hybond N ⁺ membrane. The membrane was then denatured with 0.5 M NaOH, 1.5 M NaCl and neutralized with 1.5 M NaCl, 0.5 M Tris-HCl pH 7.2, 1.0 mM EDTA pH 8.0. The DNA was bound to the membrane by UV cross-linking. The membrane was prehybridized in Church buffer (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA) for 18 h. The hybridization was carried out overnight at 65 ° C. in the same buffer Membrane washed 3 x 20 min at 65 ° C with 50 mM W2 buffer (disodium hydrogenphosphate dihydrate Na ₂ HpO ₄ .2H ₂ O) pH 7.2 and subjected to autoradiography (see Fig. 1 (B)).

It was found that 500 clones out of the 50,000 clones hybridized with the amplified DNA. 3. Characterization of clones that hybridized with DNA from cell line 578

The phage DNA was isolated from ten positive clones and subjected to a restriction cleavage with the restriction enzymes BssHII, NotI, SacII and EcoRI (cf. FIG. 2 (A), (B)). Furthermore, the gel of FIG. 2 (B) was subjected to a blot method and hybridized with the amplified DNA from the hybrid cell 578 (cf. FIG. 2 (C)).

It was shown that seven clones contained BssHII, five clones SacII and two clones NotI restriction sites.

Furthermore, 16 clones from the genomic DNA library relating to the human X chromosome, which hybridized with the amplified DNA from the hybrid cell line 578, were cleaved with EcoRI, separated on a 0.8% agarose gel, subjected to a blot method and hybridized with the amplified DNA from the hybrid cell line 578 (see FIG. 3 (A)).

It was found that bands defined in 15 clones hybridized.

The same filter was washed off and hybridized with the amplified DNA from cell line 853, which contains a human Y chromosome (see FIG. 3 (B)).

Four clones were shown to hybridize.

Three insert bands from clone X26, including the XY-common positive band, were purified from the agarose gel and hybridized in genomic Southern blots containing DNA from cell lines 578, 853 and 815 x 175 k7 (see FIG. 3 ( C), (D)).

The common positive band was found to result in smear hybridization (data not shown). The smaller band gave an X-specific hybridization signal (Fig. 3 (C), while the smallest band showed a homologous sequence between chromosomes X and Y, although different hybridization patterns were recognizable for both chromosomes (Fig. 3 (D)).

Table I

BssHIl 5'-GCGCGCNN-3 '

Notl 5'-GCGGCCGC-3 '

Sacll 5'-CCGCGGNN-3 '

Accl 5'-CGCGNNNN-3 '

Ascl 5'-GGCGCGCC-3 '

Apal 5'-GGGCCCNN-3 '

10 bps 5'-CGATCGNN-3 '

Cspl 5'-CGG (A, ηCCGN-3 '

EclXI 5'-CGGCCGNN-3 '

Fsel 5'-GGCCGGCC-3 '

Mlul 5'-ACGCGTNN-3 '

15 Nael 5'-GCCGGCNN-3 '

Narl 5'-GGCGCCNN-3 '

NciL 5'-CCNNGGNN-3 '

Nrul 5'-TCGCGANN-3 '

Rsrll 5'-CGGNCCGN-3 '

20 Sall 5'-GTCGACNN-3 '

Sfil 5'-GGCCNNGGCC-3 '

SgrAI 5'-CGCCGGCG-3 '

Srfl 5'-GCCCGGGC-3 'where N is G or C.

25

Claims

claims

1. A method for the detection of genes, comprising the following procedural steps:

(a) Ampiification of a DNA (a) with one to all of the following primers:

5'-GCGCGCNN-3 '

5'-GCGGCCGC-3 '

5'-CCGCGGNN-3 '5'-CGCGNNNN-3'

5'-GGCGCGCC-3 '

5'-GGGCCCNN-3 '

5'-CGATCGNN-3 '

5'-CGG (A, T) CCGN-3 '5'-CGGCCGNN-3'

5'-GGCCGGCC-3 '

5'-ACGCGTNN-3 '

5'-GCCGGCNN-3 '

5'-GGCGCCNN-3 '5'-CCNNGGNN-3'

5'-TCGCGANN-3 '

5'-CGGNCCGN-3 '

5'-GTCGACNN-3 '

5'-GGCCNNGGCC-3 '5'-CGCCGGCG-3'

5'-GCCCGGGC-3 'where N is G or C, and

(b) Hybridization of the amplified DNA with a DNA library.

2. The method of claim 1, wherein the DNA is a genomic DNA.

3. The method of claim 1, wherein the DNA is a cDNA.

4. The method of claim 1, wherein the DNA is an extrachromosomic DNA.

5. The method according to any one of claims 1-4, wherein the DNA library is a genomic DNA library.

6. The method according to any one of claims 1-4, wherein the DNA library is a cDNA library.

7. The method according to any one of claims 1-6, wherein the DNA and the DNA library come from the same species.

8. The method of claim 7, wherein the DNA and DNA library are from the same individual.

9. The method of claim 7, wherein the DNA and the DNA library are from different individuals.

10. The method according to any one of claims 1-6, wherein the DNA and the DNA library come from different species.

11. The method according to any one of claims 1-10, wherein different cDNAs are used for hybridization with a DNA library.

12. The method according to claim 11, wherein the expression of genes, in particular expression differences, are detected.